CN112939989B - 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine and application thereof - Google Patents
7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine and application thereof Download PDFInfo
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Abstract
The invention discloses 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]Pyrimidines and their use. 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of the pyrimidine is shown as the following formula (a), wherein R in the formula (a)1Is 3-indolyl, 4-methylphenyl, phenyl, 5- (1-methylindazolyl), 5- (1-methylindolyl), 3- (1-hydroxymethylindolyl), 3-nitro-4-methoxyphenyl or 4-methanesulfonylphenyl. The invention relates to 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The pyrimidine can effectively inhibit the proliferation of tumor cells, has strong capacity of inhibiting the aggregation of tubulin, and provides a novel tubulin inhibitor for inhibiting the proliferation of tumor cells.
Description
The technical field is as follows:
the invention belongs to the field of organic compounds, and particularly relates to a 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine compound, which can inhibit the growth of cancer cells and can be used for treating tumors.
Background art:
malignant tumor is the most serious disease endangering human health, the incidence rate of the malignant tumor is second to cardiovascular and cerebrovascular diseases, the malignant tumor is the second largest killer of human health, and the death rate of the malignant tumor even exceeds the cardiovascular and cerebrovascular diseases and is the first of all diseases. Therefore, the search and development of new drugs for treating tumors is a major issue currently facing. As microtubules play a very critical role in the proliferation and division of tumor cells, tubulin becomes an ideal target for antitumor drugs.
Microtubules play an important role in a variety of cellular processes, including spindle formation, cell shape maintenance and intracellular trafficking. The function of microtubules in cell mitosis makes them attractive targets for anticancer drugs, and microtubule targeting agents disrupt microtubule formation thereby inhibiting cancer cells from entering the G2/M phase, eventually leading to cancer cell apoptosis. Therefore, tubulin inhibitors have been widely used in the treatment of cancer.
All currently marketed tubulin inhibitors bind to the paclitaxel or vinblastine binding site in tubulin, and these compounds have the advantage of high antitumor activity and are effective against a variety of cancers. And the main problems are that: firstly, the toxic and side effects are great; secondly, drug resistance (multidrug resistance/MDR) is easy to generate; tubulin inhibitors of the colchicine binding site may overcome the above disadvantages and have therapeutic advantages over the taxane and vinca alkaloid binding sites, for example, they are better water soluble and may be administered orally; in addition, they are not susceptible to multidrug resistance. To date, a number of tubulin inhibitors based on the colchicine binding site have been found to be effective anticancer agents. Some of them have reached clinical trials, which suggests that anti-tumor drug analogs based on colchicine binding sites have great prospects for development.
Of the colchicine-based targeted tubulin inhibitors, the drugs which have been patented and put into clinical trials are listed in the attached listIn fig. 1. Cotempatin (Combretastatin) CA-4 is the most active member of the Cotempatin family isolated from the dwarf willow in south Africa. CA-4 displays strong anti-tubulin activity by binding to the colchicine site and has undergone phase II and phase III clinical studies. Replacement of the olefinic bridge of CA-4 with a carbonyl group produces phenstatin, which has similar potency and mechanism of action as CA-4. ABI-231 is an analog of CA-4, and exhibits a potent inhibitory activity against tubulin polymerization. Compd.9a is a tubulin inhibitor obtained by merging the carbonyl groups of ABI with a five-membered ring and in which one methoxy group is replaced with a selenomethyl group, which has an IC on the nanomolar scale for tumor cells50The compound has strong antitumor activity. Thus, compounds of the colchicine binding site have attracted great interest to medicinal chemists.
The invention content is as follows:
the invention aims to solve the technical problem of providing a 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine compound, which can inhibit the proliferation of tumor cells and provides a new inhibitor for inhibiting the proliferation of the tumor cells.
The scheme for solving the technical problems is as follows:
the chemical structure of the 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine is shown as the following formula (a),
(a) in the formula, R1Is 3-indolyl, 4-methylphenyl, phenyl, 5- (1-methylindazolyl), 5- (1-methylindolyl), 3-nitro-4-methoxyphenyl, 4-methylsulfonylphenyl or 3- (1-hydroxymethylindolyl).
The 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine provided by the invention is preferably one of the following compounds:
when R is1Is 3-indolyl, said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]Pyrimidine conversionThe chemical structure is as follows:
when R is1Is phenyl, said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of pyrimidine is:
when R is1Is 4-methylphenyl, said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of pyrimidine is:
when R is1Is 5- (1-methylindolyl), said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of pyrimidine is:
when R is1Is 5- (1-methylindazolyl), said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of pyrimidine is:
when R is1Is 3- (1-hydroxymethylindolyl), said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of pyrimidine is:
when R is1Is 3-nitro-4-methoxyphenyl, said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of pyrimidine is:
when R is1Is 4-methylsulfonylphenyl, said 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d]The chemical structure of pyrimidine is:
the 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine is synthesized by the following synthesis method (the reaction formula is shown as i) comprising the following steps:
firstly, carrying out nitration reaction on 4-hydroxy-3-methoxyacetophenone (compound 1) in a mixed solvent of nitric acid and acetic acid to obtain a compound 2; then under the action of dimethyl sulfate, a compound 3 is obtained; reducing under the action of palladium carbon and hydrogen to obtain a compound 4; then, diazo salt is generated, and potassium selenocyanate is added to obtain a compound 5; under the conditions of methyl iodide and sodium borohydride, a compound 6 is obtained; reacting the compound 6 with N, N-dimethylformamide dimethyl acetal to generate a compound 7; then reacting the compound 7 with 3-bromo-1H-pyrazol-5-amine in an acetic acid solvent to generate a compound 8; then the compound 8 and aromatic boric acid are subjected to Suzuki reaction to generate 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine shown in formula (a).
The reaction formula of the above method is as follows:
Reagents and conditions:(a)HNO3,AcOH,3h,r.t；(b)Dimethylsulfate,K2CO3,acetone,2d,80℃；(c)Pd/C,H2,CH3OH/THF,r.t,overnight；(d)i:HCl,H2O,NaNO2,40min,0℃；ii:NaOAc,30min,0℃；iii:KSeCN,3h,0℃-r.t；(e)NaBH4,CH3I,EtOH,10min,r.t；(f)N,N-dimethylformamide dimethyl acetal,DMF,120℃,6h；(g)3-bromo-1H-pyrazol-5-amine,AcOH,80℃,8h；(h)appropriate aryl boronic acid,Pd(dppf)Cl2,Na2CO3 aq.,DMF,95℃,12h；(i)37％HCHO aq.,NaOH aq.,3h,r.t.
experiments show that the 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine can inhibit tumor proliferation, can be used for preparing a tumor proliferation inhibitor, and has a remarkable anti-tumor effect.
Therefore, the invention also provides the application of the 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine in preparing antitumor drugs.
The invention also provides an anti-tumor medicament which comprises the 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine serving as an active ingredient.
The anti-tumor drug is preferably a drug for resisting cervical cancer, breast cancer, lung cancer or melanoma.
More preferably, the structural formula of the 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine is shown as any one of the following formulas:
preferably, the anti-tumor drug comprises 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrimidoimidazole and medically acceptable auxiliary materials.
The 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine can effectively inhibit the proliferation of tumor cells, has strong capacity of inhibiting the aggregation of tubulin, and provides a novel tubulin inhibitor for inhibiting the proliferation of the tumor cells.
Drawings
FIG. 1: tubulin inhibitors of several colchicine binding sites are listed.
FIG. 2: compound 9a inhibits tubulin aggregation in vitro.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1 Synthesis of key intermediates of the invention
(1) Synthesis of Compound 2:
8.3g of 4-hydroxy-3-methoxyacetophenone (Compound 1) was dissolved in an acetic acid solvent, and 4mL of dilute nitric acid was slowly dropped thereinto at 0 ℃ and stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was poured into ice water to precipitate out a precipitate, which was then filtered and dried to obtain compound 2 with a yield of 91.0%.1H NMR(400MHz,Chloroform-d)δ11.13(s,1H),8.32(s,1H),7.78(s,1H),4.03(s,3H),2.64(s,3H).
(2) Synthesis of Compound 3:
6.3g of Compound 2 was dissolved in acetone, and 10.4g of potassium carbonate and 4.3mL of dimethyl sulfate were added, respectively, to react at 80 ℃ for 48 hours. After the reaction is finished, ammonia water is added to remove excessive dimethyl sulfate, ethyl acetate is used for extraction, and after the solvent is concentrated, the compound 3 is obtained with the yield of 89.6%.1H NMR(400MHz,Chloroform-d)δ7.92(s,1H),7.75(s,1H),4.07(s,3H),4.01(s,4H),2.63(s,3H).
(3) Synthesis of Compound 4:
dissolving the compound 3 in a methanol solvent, adding a proper amount of palladium carbon, and reacting for 4 hours in a hydrogen environmentAfter the reaction, the solid in the solution was filtered off with celite, and the reaction solution was concentrated and subjected to column chromatography to obtain compound 4 with a yield of 75.2%.1H NMR(400MHz,Chloroform-d)δ7.04(d,J＝1.7Hz,1H),7.00(d,J＝1.6Hz,1H),3.92(s,2H),3.90(s,3H),2.55(s,2H).
(4) Synthesis of Compound 5:
adding 195mg of compound 4 into water, adding 3mL of 10% hydrochloric acid, adding 100mg of sodium nitrite into the solution under ice bath, stirring for 1 hour at 0 ℃, then adding 217mg of potassium selenocyanate, continuously stirring for 3 hours, precipitating a large amount of precipitate, filtering and drying to obtain compound 5, wherein the yield is 89.5%.1H NMR(400MHz,Chloroform-d)δ7.82(s,1H),7.58(s,1H),4.04(s,3H),3.96(s,3H),2.64(s,3H).
(5) Synthesis of Compound 6:
285mg of Compound 5 was dissolved in methanol, and 38mg of sodium borohydride was added, followed by 0.1mL of methyl iodide, and the mixture was stirred at room temperature for 10 minutes. After the reaction is finished, ethyl acetate is used for extraction, the solvent is concentrated, and then the compound 6 is obtained through column chromatography, wherein the yield is 83.5%.1H NMR(400MHz,Chloroform-d)δ7.44(s,1H),7.39(s,1H),3.93(s,4H),3.91(s,4H),2.58(s,3H),2.32(s,3H).
(6) Synthesis of compound 7:
1.0g of Compound 6 was dissolved in 15mL of ethanol, and 5.0g of N, N-dimethylformamide dimethyl acetal was added to the solution, followed by refluxing with heating and monitoring by thin layer chromatography. After the reaction was completed, the reaction solution was spin-dried, extracted with ethyl acetate-water, the ethyl acetate layer was dried over anhydrous sodium sulfate, the solution was concentrated, dichloromethane-methanol 100: column chromatography of 3 gave 1.0g of compound 7 in 79.2% yield.
The obtained compound 7 is identified by adopting nuclear magnetic resonance spectrum and mass spectrum technology, and the identification result is as follows:1H NMR(400MHz,CDCl3)δ7.80(d,J＝12.3Hz,1H),7.16(s,2H),5.63(d,J＝12.3Hz,1H),3.92(s,6H),3.89(s,3H),3.05(ss,6H).ESI-MS m/z:265.1[M+H]+266.1.
(7) synthesis of compound 8:
1.0g of Compound 7 was dissolved in 15mL of acetic acid, followed by addition of 0.61g of 3-bromo-1H-pyrazol-5-amine, heating under reflux, and monitoring by thin layer chromatography. After the reaction was completed, extraction was performed with ethyl acetate-water, the ethyl acetate layer was dried over anhydrous sodium sulfate, and the solution was concentrated, dichloromethane-methanol 100: column chromatography of 3 afforded 1.25g of compound 8. The yield thereof was found to be 91.1%.
The obtained compound 8 is identified by adopting nuclear magnetic resonance spectrum and mass spectrum technology, and the identification result is as follows:1H NMR(400MHz,CDCl3)δ8.50(d,J＝4.4Hz,1H),7.30(s,2H),6.90(d,J＝4.4Hz,1H),6.81(s,1H),3.95(s,3H),3.94(s,6H).ESI-MS m/z:363.0[M+H]+364.0.
EXAMPLE 2 Synthesis of Compound 9a
115mg of Compound 8 was dissolved in a mixed solvent of 5mL of DMF and 0.5mL of water, and 47.6mg of p-methylphenylboronic acid, 15.6mg of tetrakis (triphenylphosphine) palladium and 71.3mg of sodium carbonate were added to the above solution, heated to 95 ℃ and reacted for 10 hours, followed by thin layer chromatography. After the reaction was completed, extraction was performed with ethyl acetate-water, the ethyl acetate layer was dried over anhydrous sodium sulfate, and the solution was concentrated, dichloromethane-methanol 100: column chromatography 3 afforded 93mg of compound 9a, 78.8% yield.
Subjecting the obtained product to chemical conversionThe compound 9a is identified by adopting nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result is as follows:1H NMR(400MHz,Chloroform-d)δ8.49(d,J＝4.4Hz,1H),7.93(d,J＝8.0Hz,2H),7.76(d,J＝1.6Hz,1H),7.68(d,J＝1.6Hz,1H),7.29(d,J＝8.0Hz,2H),7.06(s,1H),6.92(d,J＝4.4Hz,1H),4.01(s,3H),3.99(s,3H),2.43(s,3H),2.37(s,2H).13C NMR(101MHz,CDCl3)δ155.8,151.8,151.2,148.7,148.5,145.5,138.9,130.0,129.4,128.1,127.3,126.3,121.3,111.6,106.6,93.3,60.1,56.1,21.3,5.1.ESI-MS m/z:[M+H]+440.1.
EXAMPLE 3 Synthesis of Compound 9b
The compound 9b was synthesized by the same method as in example 2 using the compound 8 and 5- (1-methylindole) phenylboronic acid as starting materials, and the yield was 80.2%.
The obtained compound 9b is identified by adopting nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result is as follows:1HNMR(400MHz,Chloroform-d)δ8.48(d,J＝4.3Hz,1H),8.31(s,1H),7.96(d,J＝8.5Hz,1H),7.81(s,1H),7.74(s,1H),7.41(d,J＝8.6Hz,1H),7.12(s,1H),7.10(d,J＝3.0Hz,1H),6.89(d,J＝4.3Hz,1H),6.58(d,J＝2.8Hz,1H),4.03(s,3H),4.01(s,3H),3.85(s,3H),2.40(s,3H).13C NMR(101MHz,CDCl3)δ157.3,151.8,151.3,148.5,148.5,145.3,137.2,129.6,128.7,128.1,127.5,124.2,121.3,120.4,119.3,111.6,109.4,106.2,101.6,93.0,60.1,56.1,32.9,5.2.ESI-MS m/z:[M+H]+479.1.
EXAMPLE 4 Synthesis of Compound 9c
Compound 9c was synthesized by the same method as in example 2, using compound 8 and 5- (1-methylindazole) phenylboronic acid as starting materials, and the yield was 81.6%.
The obtained compound 9c adopts nuclear magnetic resonance spectrum and mass spectrum technologyAnd (4) carrying out identification, wherein the identification result is as follows:1H NMR(400MHz,Chloroform-d)δ8.50(d,J＝4.4Hz,1H),8.38(s,1H),8.12(d,J＝8.8,1H),8.07(s,1H),7.75(d,J＝1.9Hz,1H),7.70(d,J＝1.9Hz,1H),7.48(d,J＝8.8Hz,1H),7.12(s,1H),6.92(d,J＝4.4Hz,1H),4.13(s,3H),4.02(s,3H),4.00(s,3H),2.38(s,3H).13C NMR(101MHz,CDCl3)δ156.1,151.8,151.3,148.8,148.6,145.5,140.1,133.4,128.2,127.3,125.6,125.2,124.3,121.3,119.2,111.5,109.2,106.6,93.2,60.2,56.1,35.6,5.1.ESI-MS m/z:[M+H]+480.1.
EXAMPLE 5 Synthesis of Compound 9d
The synthesis of compound 9d is divided into 2 steps, the first step: the compound 8 and 1- (benzenesulfonyl) -3-indolylboronic acid were used as starting materials in the same manner as in example 2 with a yield of 88.7%; the second step is that: dissolving the product obtained in the first step in a mixed solvent of ethanol and water, adding a proper amount of sodium hydroxide, and hydrolyzing to obtain a compound 9d with the yield of 78.4%.
The obtained compound 9d is identified by adopting nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result is as follows:1HNMR(400MHz,Chloroform-d)δ8.69(s,1H),8.49(d,J＝4.4Hz,1H),8.44(d,J＝8.5Hz,1H),7.83(d,J＝1.5Hz,1H),7.74(s,1H),7.68(d,J＝1.6Hz,1H),7.41(d,J＝8.1Hz,1H),7.29-7.23(m,2H),7.03(s,1H),6.88(d,J＝4.4Hz,1H),4.03(s,3H),4.00(s,3H),2.36(s,3H).13C NMR(101MHz,CDCl3)δ152.7,151.9,150.7,148.7,148.5,145.4,136.5,128.0,127.8,125.4,124.0,122.7,121.5,121.4,120.7,111.6,111.3,110.4,106.0,60.2,56.1,5.2.ESI-MS m/z:[M+H]+465.1.
EXAMPLE 6 Synthesis of Compound 9e
The compound 9e was synthesized by the same method as in example 2 using the compound 8 and phenylboronic acid as starting materials, and the yield was 82.9%.
The obtained compound 9e is identified by adopting nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result is as follows:1H NMR(400MHz,Chloroform-d)δ8.52(d,J＝4.2Hz,1H),8.04(d,J＝7.1Hz,2H),7.76(d,J＝1.9Hz,1H),7.68(d,J＝1.9Hz,1H),7.48(t,J＝7.3Hz,2H),7.42(t,J＝7.3Hz,1H),7.10(s,1H),6.94(d,J＝4.4Hz,1H),4.02(s,3H),4.00(s,3H),2.38(s,3H).13C NMR(101MHz,CDCl3)δ155.7,151.8,151.2,148.9,148.6,145.6,132.8,129.0,128.7,128.2,127.3,126.4,121.3,111.6,106.8,93.6,60.1,56.1,5.1.ESI-MS m/z:[M+H]+426.1.
EXAMPLE 7 Synthesis of Compound 9f
The synthesis method of the compound 9f used the compound 8 and 3-nitro-4-methoxyphenylboronic acid as raw materials, and the method was the same as in example 2, with a yield of 83.5%.
The obtained compound 9f is identified by adopting nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result is as follows:1H NMR(400MHz,Chloroform-d)δ8.57(s,1H),8.54(d,J＝4.3Hz,1H),8.15(d,J＝8.7Hz,1H),7.75(s,1H),7.65(s,1H),7.20(d,J＝8.7Hz,1H),7.05(s,1H),6.98(d,J＝4.3Hz,1H),4.04(s,3H),4.02(s,3H),4.01(s,3H),2.38(s,3H).13C NMR(101MHz,CDCl3)δ153.3,153.1,151.9,151.4,149.2,148.8,145.6,139.8,131.9,128.4,126.9,125.8,123.7,121.2,113.7,111.5,107.1,93.3,60.2,56.7,56.1,5.2.ESI-MS m/z:[M+H]+501.1.
EXAMPLE 8 Synthesis of 9g Compound
The compound 9g was synthesized by the same method as in example 2 using the compound 8 and 4-methanesulfonylphenylboronic acid as starting materials, and the yield was 85.6%.
Will be describedThe obtained 9g of compound is identified by adopting nuclear magnetic resonance spectrum and mass spectrum technology, and the identification result is as follows:1HNMR(400MHz,Chloroform-d)δ8.56(d,J＝4.1Hz,1H),8.21(d,J＝8.2Hz,2H),8.04(d,J＝8.2Hz,2H),7.69(s,1H),7.65(s,1H),7.17(s,1H),7.00(d,J＝4.1Hz,1H),4.02(s,3H),3.98(s,3H),3.11(s,3H),2.36(s,3H).13C NMR(101MHz,CDCl3)δ153.4,151.9,151.3,149.4,148.8,145.9,140.4,138.2,128.4,127.9,127.1,126.8,121.2,111.5,107.6,94.6,60.2,56.1,44.5,5.1.ESI-MS m/z:[M+H]+504.0.
EXAMPLE 9 Synthesis of Compound 9h
Dissolving 50mg of the compound 9d in an ethanol solvent, adding 1mL of a formaldehyde solution and 1mL of a 10% sodium hydroxide solution, stirring at room temperature for 4 hours, extracting with ethyl acetate after the reaction is finished, concentrating the solvent, and performing column chromatography to obtain the compound 9h, wherein the yield is 80.6%.
The obtained compound 9h is identified by adopting nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result is as follows:1HNMR(400MHz,Methanol-d4)δ8.41(d,J＝4.5Hz,1H),8.36(d,J＝7.8Hz,1H),7.81(s,1H),7.76(d,J＝1.7Hz,1H),7.67(d,J＝1.7Hz,1H),7.55(d,J＝8.1Hz,1H),7.28(t,J＝7.3Hz,1H),7.21(t,J＝7.4Hz,1H),6.97(s,1H),6.90(d,J＝4.5Hz,1H),5.63(s,2H),3.98(s,3H),3.97(s,3H),2.33(s,3H).13C NMR(101MHz,CD3OD)δ152.9,151.8,150.2,148.6,148.4,145.9,136.4,128.1,127.6,127.5,126.4,122.6,121.5,121.3,120.8,111.5,109.9,109.5,106.0,92.3,69.3,60.0,55.9,4.8.ESI-MS m/z:[M+H]+495.1.
example 107- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidines for investigating the Effect on tumor cell inhibition
The tumor-inhibiting effect of the compounds of the present invention was demonstrated by the following test methods.
These effects indicate that the compounds of the present invention have a significant tumor cell inhibitory effect and are useful for the treatment of cancer. The specific test method is as follows:
first, experimental purpose and principle
Purpose of the experiment: MTT method is adopted to detect the inhibiting effect of a series of synthesized 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine compounds on the proliferation of tumor cells.
The experimental principle is as follows: MTT colorimetry is a method for detecting survival and growth of cells, and its principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan, which is deposited in cells, while dead cells lack this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in living cells, an enzyme linked immunosorbent assay detector is used for detecting an absorbance value (OD value) under 570nM, the quantity of the living cells can be reflected according to the absorbance value, and in a certain range, the smaller the OD value is, the weaker the cell activity is, and the better the proliferation inhibition effect of the drug is.
Second, basic information of reagent
Third, reagent preparation
1. RPMI-1640 complete medium
Preparing 1L of RPMI-1640 culture medium, taking a corresponding amount of RPMI-1640 powder, dissolving in a beaker containing 800ml of triple distilled water, and stirring for 4 hours until the powder is completely dissolved. 2g of NaHCO were added3And stirring until the mixture is completely dissolved. Adjusting the pH value with 1mol/L hydrochloric acid to 7.2-7.4, and metering to 1L. Filtering with a filter membrane with pore diameter of 0.22 μm, filtering with high pressure filter, packaging, and storing at 4 deg.C. When in use, 5% of serum is added to form a complete culture medium, and the culture medium can be used for cell culture.
2、MTT
Wrapping 50ml of centrifuge tube with tinfoil paper in dark place, precisely weighing 250mg of MTT powder, adding into centrifuge tube, adding 50ml of PBS to completely dissolve MTT powder, filtering with 0.22 μm filter membrane for sterilization, subpackaging, and storing at-20 deg.C in dark place.
3. Compound configuration
Autoclaved EP tubes were used to weigh the compounds, and corresponding amounts of DMSO were added to the EP tubes to bring the solution to a 100mM stock and diluted to 30mM, 10mM, 3mM, 1mM respectively. When in use, the culture medium is diluted by 1000 times with a corresponding amount of the culture medium, and working solution with the concentration of 0.1 mu M, 0.3 mu M, 1 mu M, 10 mu M, 30 mu M and 100 mu M can be prepared.
Fourth, the experimental process
(1) Collecting cervical cancer cell (Hela), breast cancer cell (MCF-7), lung cancer cell (A549), and melanoma cell (B16-F10) in logarithmic growth phase, digesting, and adjusting cell number concentration to 2.5 × 104one/mL, 100. mu.l/well into 96-well plates. At 37 ℃ 5% CO2Culturing in a cell culture box overnight until the cells adhere to the wall.
(2) The original culture medium was aspirated, and different concentrations of 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine series compounds were added to each group, with each compound having a gradient concentration of 0.1. mu.M, 0.3. mu.M, 1. mu.M, 10. mu.M, 30. mu.M, 100. mu.M, 3 replicates per treatment. The culture was continued for 72h in a cell culture incubator with 0.1% DMSO as a control, Paclitaxel (PTX) as a positive control, and no cells and compound as a blank control.
(3) Mu.l of MTT solution was added to each well and incubated for 4h in an incubator.
(4) The medium was discarded, 100. mu.l of DMSO was added to each well, and formazan crystals were sufficiently dissolved by shaking for 15 min.
(5) The absorbance at 570nm was measured using an enzyme linked immunosorbent assay.
(6) The cell growth inhibition rate was calculated according to the following formula:
the inhibition rate is [ (As-Ab)/(Ac-Ab) ]. times.100%
As: absorbance of assay well (cell, MTT, Compound)
Ac: absorbance of control wells (cell, MTT, no Compound)
Ab: absorbance of blank wells (cell and Compound free, MTT containing)
The results are shown in Table 1 below.
TABLE 1 half inhibitory concentration of the compound on tumor cell growth
As can be seen from Table 1, the 7- (3, 4-dimethoxy-5-selenomethylphenyl) -pyrrolo [2,3-d ] pyrimidine series compounds can effectively inhibit the growth of tumor cells, and particularly, the compound 9a has stronger inhibition effect on Hela cells and B16-F10 cells than positive control PTX. The inhibitory effect of compound 9b on a549 cells was stronger than that of the positive control PTX.
Example 11 inhibition of tubulin aggregation in vitro
Compound 9a was tested for its effect on tubulin polymerization in need of assay using a fluorescence-based tubulin polymerization assay kit (Cat. # BK011P, cytosketon, inc., USA) according to the manufacturer's protocol. Tubulin was suspended in ice-cold G-PEM buffer (80mM PIPES, 2mM MgCl)20.5mM EGTA, 1mM GTP, 20% (v/v) glycerol) and added to a 96-well plate containing the indicated concentration of drug or blank. Polymerization of tubulin was monitored at 37 ℃ for 90min at 1-minute intervals with a microplate reader (FASCalibur, BD Biosciences, USA), the absorbance value was converted into the inhibition rate of polymerization of microtubules, and IC was calculated with SPSS software50The value is obtained. The results are shown in FIG. 2, from which FIG. 2 it can be seen that IC for compound 9a50The value was 2.4uM, therefore compound 9a was able to inhibit tubulin aggregation in vitro.
Claims (5)
1. A compound of the formula:
2. the use of a compound of claim 1 in the preparation of an anti-neoplastic drug, said anti-neoplastic drug being a drug against cervical cancer, breast cancer or melanoma.
3. An antitumor agent characterized by comprising the compound according to claim 1 as an active ingredient, wherein the antitumor agent is an agent against cervical cancer, breast cancer or melanoma.
4. The antitumor agent as claimed in claim 3, wherein the antitumor agent comprises the compound as claimed in claim 1 and a pharmaceutically acceptable adjuvant.
5. A method of synthesizing the compound of claim 1, comprising the steps of:
the reaction formula is as follows:
firstly, carrying out nitration reaction on a compound 1 in a mixed solvent of nitric acid and acetic acid to obtain a compound 2; then under the action of dimethyl sulfate, a compound 3 is obtained; reducing under the action of palladium carbon and hydrogen to obtain a compound 4; then, diazo salt is generated, and potassium selenocyanate is added to obtain a compound 5; under the conditions of methyl iodide and sodium borohydride, a compound 6 is obtained; reacting the compound 6 with N, N-dimethylformamide dimethyl acetal to generate a compound 7; then reacting the compound 7 with 3-bromo-1H-pyrazol-5-amine in an acetic acid solvent to generate a compound 8; then subjecting compound 8 to a Suzuki reaction with an aromatic boronic acid to produce the compound of claim 1;
said R1Is 5- (1-methylindolyl).
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| PCT/CN2021/125116 WO2022033612A1 (en) | 2021-03-03 | 2021-10-21 | 7-(3,4-dimethoxy-5-selenomethylphenyl)-pyrrolo[2,3-d]pyrimidine and use thereof |
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